Intellectual Merit: It has become increasingly clear that transcriptional and post-transcriptional steps of gene expression are coupled, interdependent and potentially co-regulated. Much of the evidence for regulating mRNA processing through coupling with transcription is derived from artificial systems. While these models demonstrate the capacity for regulating post-transcriptional steps of gene expression through interactions with transcriptional machinery, there are few natural examples of where this occurs in vivo. Recently, the Zinc fingers and homeoboxes 2 (Zhx2) gene was cloned and shown to be responsible, in part, for repressing the expression of alpha-fetoprotein (AFP) and other genes in the liver after birth. Published studies have shown that the AFP promoter is sufficient to confer Zhx2 regulation on a heterologous reporter gene. However, preliminary results have shown that the transcription rate across the AFP gene is the same in the presence or absence of Zhx2, even though AFP mRNA accumulation is repressed. Moreover, this repression occurs because splicing of multiple AFP introns is inhibited. Because Zhx2 functions in a promoter-dependent manner but acts at a post-transcriptional level, this system provides a unique opportunity to understand a biologically relevant and novel mechanism that couples transcriptional to post-transcriptional gene regulation. Based on preliminary studies, it is hypothesized that Zhx2 acts through the promoter of its target genes to decrease the splicing efficiency of the nascent RNA and thus reduce fully processed mRNA levels. To test this hypothesis and investigate mechanistic details, mice that express a FLAG-tagged Zhx2 transgene, but low levels of endogenous Zhx2, have been generated. Using these mice, this project will determine whether Zhx2 is a direct or indirect regulator of AFP expression and whether it associates with target promoters or along the whole gene by using chromatin immunoprecipitation (ChIP) assays. Combining ChIP with DNA sequencing will identify other Zhx2-regulated genes. The mechanism of AFP RNA splicing repression will be investigated by comparing the pol II complex in the presence or absence of Zhx2 by ChIP. Whether splicing repression occurs co-transcriptionally will be determined by measuring splicing of nascent RNA. Finally, this project will identify proteins that interact with Zhx2, which will provide essential information about Zhx2 function. The components of FLAG-Zhx2-containing protein complexes from adult mouse liver will be identified by mass spectrophotometry. The long-term goal of the project is to understand Zhx2-mediated regulation of AFP because it is a novel regulatory mechanism that appears to couple transcription to post-transcriptional events in the liver. Details of this mechanism will lead to a better understanding of gene regulation at the step of RNA biogenesis.
Broader impacts: Graduate, undergraduate, and, potentially, high school students will be involved in the research, building on the laboratory's strong history of student training. Results of the research will be shared with the broader scientific community through national meeting presentations and publications. Scientifically, the research has the potential to uncover novel insights into a poorly understood mechanism of gene regulation.
